A sensitive and specific two stage polymerase chain reaction (PCR) method was devised for the simultaneous amplification and detection of genomic sequences of hepatitis B virus (HBV) and hepatitis C virus (HCV) in serum. RNA and DNA were extracted from sera. HCV-RNA was first reverse transcribed to cDNA. DNA sequences from both HCV (286 bp) and HBV (128 bp) were co-amplified by using primer pairs derived from conserved regions. PCR products were separated by agarose gel electrophoresis and their molecular size determined. Specificity of the PCR products for HBV and HCV sequences was confirmed by Southern blot analysis with 32P-end labeled oligonucleotide probes. Independent sets of sera, positive and negative by PCR for either HBV-DNA or HCV-RNA, were used as controls. Nine donor sera, antibody positive for both HBV (anti-HBc/HBs) and HCV (anti-c100-3) were tested. Sera from 5 patients with elevated alanine amino transferase (ALT) but negative for antibodies to HBV and HCV were also tested. Our assay detected HBV- and HCV-specific genomic sequences in 9/9 sera reactive for both HBV and HCV antibodies. Sera from patients with elevated ALT and antibody negative were all PCR positive for HCV-RNA, but negative for HBV-DNA. These results demonstrate that sera containing HBV and HCV sequences can be co-amplified by PCR and the products detected by agarose gel electrophoresis. Since this method is non-isotopic, it may be particularly useful in the clinical laboratory setting.